1. Field of the Invention
The present invention relates to isolated polypeptides having lysophospholipase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
2. Description of the Related Art
Phospholipases are enzymes that participate in the hydrolysis of phospholipids which consist of a glycerol backbone with two fatty acids in an outer (sn-1) and the middle (sn-2) positions and esterified with phosphoric acid in the third position. The phosphoric acid may, in turn, be esterified to an amino alcohol.
Several types of phospholipase activity can be distinguished which hydrolyze the fatty acyl moieties. Phospholipase A1 and A2 catalyze the deacylation of one fatty acyl group in the sn-1 and sn-2 positions, respectively, from a diacylglycerophospholipid to produce lysophospholipid. Lysophospholipase (also called phospholipase B by the Nomenclature Committee of the International Union of Biochemistry on the Nomenclature and Classification of Enzymes {Enzyme Nomenclature, Academic Press, New York, 1992}) catalyzes the hydrolysis of the remaining fatty acyl group in a lysophospholipid. A phospholipase B has been reported from Penicillium notatum (Saito et al., 1991, Methods in Enzymology 197:446-456) which catalyzes the deacylation of both fatty acids from a diacylglycerophospholipid and intrinsicly possesses lysophospholipase activity.
Fungal enzymes with phospholipase activity have been reported from various sources, including Cryptococcus neoformans (Chen et al., Infection and Immunity 65: 405-411), Fusobacterium necrophorum (Fifis et al., 1996, Veterinary Microbiology 49: 219-233), Penicillium notatum (also known as Penicillium chrysogenum; Kawasaki, 1975, Journal of Biochemistry 77: 1233-1244; Masuda et al., 1991, European Journal of Biochemistry 202: 783-787), Penicillium cyclopium (Mustranta et al., 1995, Process Biochemistry 30: 393-401), Saccharomyces cerevisia (Ichimasa et al., 1985, Agric. Biol. Chem. 49: 1083-1089; Paultauf et al., 1994, Journal of Biological Chemistry 269: 19725-19730), Torulaspora delbrueckii (old name Saccharomyces rosei, Kuwabara, 1988, Agric. Biol. Chem. 52: 2451-2458; Watanabe et al., 1994, FEMS Microbiological Letters 124: 29-34), Schizosaccharomyces pombe (Oishi et al., 1996, Biosci. Biotech. Biochem. 60: 1087-1092), Neurospora crassa (Chakravarti et al., 1981, Archives of Biochemistry and Biophysics 206: 393-402), Aspergillus niger (Technical Bulletin, G-zyme(trademark) G6999, Enzyme Bio-Systems Ltd.; Mustranta et al., 1995, supra), Corticium centrifugum (Uehara et al., 1979, Agric. Biol. Chem. 43: 517-525), Fusarium oxysporum (WO 98/26057), and Fusarium solani (Tsung-Che et al., 1968, Phytopathological Notes 58:1437-38).
Fungal phospholipase genes have been cloned from several sources including Penicillum notatum (Masuda et al., 1991, supra), Torulaspora delbrueckii (Watanabe et al., 1994, FEMS Microbiology Letters 124: 29-34), Saccharomyces cerevisiae (Lee at al., 1994, Journal of Biological Chemistry 269: 19725-19730), Aspergillus (JP 10155493), Neurospora crassa (EMBL 042791), and Schizosaccharomyces pombe (EMBL 013857).
It is an object of the present invention to provide improved polypeptides having lysophospholipase activity and nucleic acid encoding the polypeptides.
The present invention relates to isolated polypeptides having lysophospholipase activity selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 65% identity with amino acids 38 to 654 of SEQ ID NO. 2 or amino acids 17 to 648 of SEQ ID NO. 16;
(b) a polypeptide encoded by a nucleic acid sequence which hybridizes under high stringency conditions with (i) nucleotides 214 to 2061 of SEQ ID NO. 1 or nucleotides 49 to 1944 of SEQ ID NO. 15; (ii) the genomic DNA sequence containing nucleotides 214 to 2061 of SEQ ID NO. 1 or nucleotides 49 to 1944 of SEQ ID NO. 15; (iii) a subsequence of (i) or (ii) of at least 100 nucleotides; or (iv) a complementary strand of (i), (ii), or (iii);
(c) an allelic variant of (a) or (b); and
(d) a fragment of (a), (b) or (c), wherein the fragment has lysophospholipase activity.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.